MBD2 (methyl-CpG-binding domain protein 2) is a member of the MBD protein family. MBD2 selectively binds to methylated DNA and suppresses transcription from a methylated target gene through recruiting transcriptional repressor complexes, which contain Mi-2/NuRD or HDACs. MBD2 has also been shown to catalyze demethylation by directly removing methyl groups from 5-methylcytosine residues in DNA. MBD2 is demonstrated to be associated with tumorigenesis. For example, deficiency of MBD2 suppresses intestinal tumor formation, indicating that MBD2 is not necessary only for tumor development, but also for tumor growth. The binding activity of MBD2 to methylated DNA may be affected by MBD2 mutation and by biochemical or pharmacological intervention. So far, there are few assays available for measuring MBD2 binding activity in vitro. The EpiQuik™ MBD2 Binding Activity Assay Kit provides a unique procedure to measure binding activity of MBD2 to methylated DNA. The kit has following features:

The EpiQuik™ MBD2 Binding Activity Assay Kit is designed for measuring in vitro or intracellular MBD2 binding activity. In an assay with this kit, activated MBD2 protein contained in nuclear extract binds to methylated DNA and forms the MBD2/methylated DNA complex. The complex is then captured to a specificallytreated strip well plate. MBD2 is recognized with a specific antibody and measured colorimetrically.

PRODUCT USE INFORMATION

The EpiQuik™ MBD2 Binding Activity Assay Kit is suitable for measuring MBD2 binding activity in human cells/tissues.
Epigentek guarantees the performance of all products in the manner described in our product instructions.
Epigentek reserves the right to change or modify any product to enhance its performance and design.
The EpiQuik™ MBD2 Binding Activity Assay Kit is for research use only and is not intended for diagnostic or therapeutic application.
EpiQuik™ is a trademark of Epigentek Group Inc.
The EpiQuik™ MBD2 Binding Activity Assay Kit and method of use are covered by a pending US patent.

* For maximum recovery of the products, centrifuge the original vial after thawing prior toopening the cap.

SHIPPING AND STORAGE
The kit is shipped in two parts: one part at ambient room temperature, and the second part on frozen ice packs at 4°C. Upon receipt: (1) Store MB5 at –20°C away from light; (2) Store MB1, MB3, MB4, MB6 and 8-Well Assay Strips at 4°C away from light; and (3) Store all other components (MB2, MB7) at room temperature. The kit is stable for up to 6 months from the shipment date, when stored properly.

Note: Check if wash buffer, MB1, contains salt precipitates before using. If so, warm (at room temperature or 37°C) and shake the buffer until the salts are redissolved.

PROTOCOL
1. Prepare nuclear extracts from treated and untreated cells or tissues by using you own successful method. For your convenience and the best results, Epigentek offers the EpiQuik™ Nuclear Extraction Kit (Cat. No. OP-0002-1) optimized for use in the EpiQuik™ series. Nuclear extracts can be used immediately or stored at -80°C for future use.

2. Determine the number of strip wells required. Leave these strips in the plate frame (remaining unused strips can be placed back in the bag. Seal the bag tightly and store at 4°C). Dilute with distilled water (pH 7.2 to 7.5) at a 1:10 ratio (e.g., 1 ml of GU1 + 9 ml of distilled water). Wash the strip wells once with 150 μl of the diluted MB1.

3. Add 26 μl of MB2, 1 μl of MB3, and then add 3 μl of nuclear extracts (4-20 μg) or purified MBD2 protein to each strip well. Mix and cover the strip wells with Parafilm M and incubate at 37°C for 60 minutes. For the blank, add 3 μl of MB2 instead of nuclear extracts.

4. Aspirate and wash each well with 150 μl of diluted MB1 three times.

5. Dilute MB4 (at a 1:100 ratio) to 1 μg/ml with diluted MB1. Add 50 μl of the diluted MB4 to each strip well and incubate at room temperature for 60 minutes on an orbital shaker (50-100 rpm).
6. Aspirate and wash each well with 150 μl of diluted MB1 four times.

7. Dilute MB5 (at a 1:1000 ratio) to 1 μg/ml with diluted MB1. Add 50 μl of the diluted MB5 to each strip well and incubate at room temperature for 30 minutes.

8. Aspirate and wash each well with 150 μl of diluted MB1 four times. Allow 3 minutes for last wash.

9. Add 100 μl of MB6 to each well and incubate at room temperature for 2-10 minutes away from light. Monitor the color development in the sample and control wells (blue).

10. Add 50 μl of MB7 to each well to stop enzyme reaction when the color in the standard wells containing the higher concentrations of standard control turns medium blue. The color should change to yellow and absorbance can be read on a microplate reader at 450 nm within 2-15 min

11. Calculate % binding of MBD2:

TROUBLESHOOTING
No Signal for the Sample
High Background Present for the Blank

291-58601 Alkaline Phospatase (ALP) Assay Alkaline Phosphatase (ALP) is distributed in a variety of tissues such as liver, bone, and small intestine in animals. The change of the enzyme activity in tissues is a B-Bridge 900 tests 233.1